The chromophore p-nitrophenol is extensively used in reagents designed to determine the presence and/or concentration of specific enzymes in test samples containing same. In spectrophotometric assays employing such reagents, the enzyme concentration is determined by the formula EQU enzyme units/liter=.DELTA.A/minute X(1/.epsilon.).times.F
where .epsilon. is the molar absorptivity of p-nitrophenol and F is a function of sample and reagent volumes and pathlength. The expression (1/.epsilon.).times.F is referred to as the calculation factor.
Within the pH range of 5 to 10 the molar absorptivity of p-nitrophenol varies with temperature and therefore the calculation factor varies with temperature. This variation necessitates (1) recalculation of the factor for each assay temperature employed; and (2) stringent maintenance of the selected temperature during assay.
Applicant overcomes the foregoing disadvantages by providing assay reagents wherein the temperature dependence of the chromophore is substantially eliminated.